Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

Secretion of unprocessed human surfactant protein B in milk of transgenic mice

Because of the apparent clinical importance of human pulmonary surfactant B (SP-B), the expression of SP-B was directed to the mammary gland of transgenic mice using previously characterized rat whey acidic protein (WAP) regulatory sequences. rWAP/SP-B mRNA was expressed specifically in the mammary gland, and ranged from 1 to 5% of the endogenous WAP mRNA levels. SP-B was detected immunologically in both tissue and milk. The transgene product had an apparent molecular weight of 40--45 kDa, corresponding to the predicted size of the SP-B proprotein. Incubation of an SP-B-enriched fraction of milk with cathepsin D in vitro produced 20--25 kDa species, consistent with cleavage of the amino terminal domain by cathepsin D. This was confirmed using antibodies specific to the carboxy-terminal domain of SP-B. However, the appearance of only the SP-B proprotein in milk suggests that cathepsin D is not involved in the in vivo processing of SP-B. The SP-B proprotein can be expressed in milk of transgenic mice without any observed effects on mammary gland morphology or lactation     

Some applications of reversed-phase high-performance liquid chromatography to oligosaccharide separations

Oligosaccharide separation on reversed-phase high-performance liquid chormatographic columns have been examined using a range of aqueous solvents. Addition of anionic, cationic and non-ionic surfactants, tetramethyl urea and organic solvents to the mobile phase cause faster elution of oligosaccharides, and allow the separation of the larger oligomers in an acceptable time. Addition of neutral, inorganic salts increase the retention factors considerably, and allows good resolution of some compounds poorly resolved in water alone.The mechanism operating in the separations approximates to that invoked in the solvophobic theory of reversed-phase chromatography. There is some evidence also of hydrogen bond effects. The improvements described should prove useful in the isolation and analysis of neutral oligosaccharides in general, and in structural analyses of polysaccharides in paritcular.     

Solubilization of the nitrendipine receptor from skeletal muscle transverse tubule membranes. Interactions with specific inhibitors of the voltage-dependent Ca2+ channel

The nitrendipine receptor associated with the voltage-dependent calcium channel from rabbit skeletal muscle transverse tubule membranes has been solubilized by detergent extraction. A highly stable solubilized receptor preparation was obtained using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate as detergent with phospholipids or glycerol present as stabilizing agents. Binding of [3H]nitrendipine to the solubilized receptor was reversible and saturable. At 4 degrees C the equilibrium dissociation constant of the [3H]nitrendipine X receptor complex was 7 +/- 3 nM and was close to that determined from the rate constants of association (k1 = 1.3 10(5) M-1 s-1) and dissociation (k-1 = 1.10 X 10(-3) s-1) of 8.4nM. The nitrendipine concentration that gave a half-maximal inhibition of [3H]nitrendipine binding to the solubilized receptor was 10 nM, which was similar to the values for the dissociation constant determined for the radiolabelled ligand. [3H]Nitrendipine binding to its solubilized receptor was also inhibited by other antiarrythmic drugs, such as bepridil and verapamil, and enhanced by d-cis-diltiazem. Since these drugs are apparent non-competitive inhibitors of [3H]nitrendipine binding it was concluded that these different binding sites are tightly coupled. Sucrose density sedimentation of solubilized nitrendipine receptor resulted in the separation of three [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4 S, 14.4 S and 21 S.     

Biological activity assessment of the 26,23-lactones of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 and their binding properties to chick intestinal receptor and plasma vitamin D binding protein

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.     

Vasoactive properties of urotensins I and II in a columbiform and three galliform birds, and a bioassay for urotensin I

Summary Crude extracts ofGillichthys urophyses and chromatographically purified urotensin I (UI) and urotensin II (UII) (fromCatostomus urophyses) were injected intravenously intoCoturnix coturnix japonica, Colinus virginianus, Alectoris graeca (Galliformes) andColumba livia (Columbiformes). Changes in arterial blood pressure were monitored. UI elicited dose-dependent vasodepressor responses in all birds. Thioglycollate treatment abolished the depressor action of arginine vasotocin but not that of UI (in all birds). UII was a pressor agent inCoturnix andColinus, both members of the Galliformes. However, the hormone had no pressor activity inColumba, a member of the Columbiformes, and in another member of the Galliformes,Alectoris. The pressor effect of UII was also dose-dependent. Injection of crude urophysial extract intoColinus andCoturnix, therefore, elicited biphasic responses. UII effects can be abolished by prior incubation with carboxypeptidase A. Intravenous injection of the -adrenoceptor blocker, phenoxybenzamine, prior to urotensin injections had no effect on the responses to urotensins. As the chukar,Alectoris graeca, is sensitive to UI and resistant to anesthesia and surgery, and does not readily develop tachyphylaxis to repeated UI injections, its use is recommended as a routine bioassay animal for UI.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland     

Checklist of the species of the aseptate Gregarine family Urosporidae

A checklist is given of the 45 named species of the family Urosporidae (phylum Protozoa, subphylum Apicomplexa, class Sporozoa, subclass Gregarinia, order Eugregarinida, suborder Aseptatina) together with their synonyms, the names of their hosts, their locations in the hosts, their known geographic distribution, and key references. Another list is given of synonyms, lapsi calami, nomina nuda, etc. associated with the genera of this family. The following taxonomic-nomenclatural innovations are introduced—NEW NAME: Gonospora good-richae nom. nov. in the polychaete Arenicola ecaudata; NEW COMBINATIONS: Urospora grassei (Changeux, 1961) in the sea cucumbers Holothuria spp.; Urospora schneideri (Mingazzini, 1891) in the sea cucumbers Holothuria spp; Gonospora gonadipertha (Djakonov, 1923) in the sea cucumber Cucumaria frondosa; Gonospora stichopi (Lützen, 1967) in the sea cucumber Stichopus tremulus.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.